Microbial Profiling using Mothur pipeline

…BRIEF INTRO IN PROGRESS…


Bioinformatics Analysis Snakemake workflow

A tentative snakemake workflow that defines mothur bioinformatics rules in a DAG (directed acyclic graph) format. A detailed interactive snakemake report is available here.


Getting started

Create a mothur YAML file

name: mothur
channels:
    - conda-forge
    - bioconda
    - defaults
dependencies:
    - mothur =1.48.0
    - vsearch =2.22.1

Create mothur env using the YAML file

conda activate base
conda env create -n mothur48 --file mothur48.yml
conda activate mothur48

Download references databases

  • Download Silva alignment reference database.
  • Process degapped Silva classifier from Silva alignments.
  • Download RDP classifier.

Example: Downloading mothur references using mothurReferences.sh script on CLI.

bash workflow/scripts/mothurReferences.sh


Mothur classification methods

There are four methods that can be used to profile microbial communities present in a sample. Here we briefly decribe each method:

1.Classify OTUs

  • OTUs (Operational Taxonomic Units (OTUs)) are clusters of similar sequences and are commonly accepted as analytical units in microbial profiling when using 16S rRNA gene markers.

2. Classify Phylotypes

  • A phylotype in microbiome research is a DNA sequence or group of sequences sharing more than an arbitrarily chosen level of similarity of a 16S rRNA gene marker.

3. Classify ASVs

  • ASVs Amplicon Sequence Variants (ASVs)in microbiome research is any inferred single DNA sequences recovered from a bioinformatics analysis of 16S rRNA marker genes.
  • ASV is typically really a cluster of sequences that are one or two bases apart from each other.

4. Classify Phylogenies

  • Microbial phylogenies are from gene sequence homologies. Models of mutation determine the most-likely evolutionary histories.


Preliminary OTU analysis using Mothur

The preliminary analysis (alpha_beta_diversity rule) is part of the bioinformatics analysis. It includes:

  • Creating reads count for each group.
  • Subsampling for downstream analysis.
  • Rarefaction.
  • Computing Alpha diversity metrics.
  • Computing Beta diversity metrics.
  • Getting sample distances.
  • Constructing sample phylip tree.
  • Generating ordination matrices including PCoA and NMDS.


#!/bin/bash

conda activate base wget https://data.qiime2.org/distro/core/qiime2-2023.2-py38-osx-conda.yml conda env create -n qiime2-2023.2 –file qiime2-2023.2-py38-osx-conda.yml conda activate qiime2-2023.2 qiime info


Microbial Profiling using QIIME 2 pipeline

Get QIIME2 YAML file

wget https://data.qiime2.org/distro/core/qiime2-2023.2-py38-osx-conda.yml

Create qiime2 env and install qiime2

Current YAML file: qiime2-2023.2-py38-osx-conda.yml available

The qiime2 YAML file contains over 500 dependencies. Listed below is just a few QIIME 2 framework dependencies to get the installation started.

name: qiime2202320
channels:
    - qiime2/label/r2023.2
    - conda-forge
    - bioconda
    - defaults
dependencies:
    - q2cli=2023.2.0
    - qiime2=2023.2.0
    - python=3.8.16
    - q2-alignment=2023.2.0
    - q2-composition=2023.2.0
    - q2-cutadapt=2023.2.0
    - q2-dada2=2023.2.0
    - q2-deblur=2023.2.0
    - q2-demux=2023.2.0
    - q2-diversity=2023.2.0
    - q2-diversity-lib=2023.2.0
    - q2-emperor=2023.2.0
    - q2-feature-classifier=2023.2.0
    - q2-feature-table=2023.2.0
    - q2-fragment-insertion=2023.2.0
    - q2-gneiss=2023.2.0
    - q2-longitudinal=2023.2.0
    - q2-metadata=2023.2.0
    - q2-mystery-stew=2023.2.0
    - q2-phylogeny=2023.2.0
    - q2-quality-control=2023.2.0
    - q2-quality-filter=2023.2.0
    - q2-sample-classifier=2023.2.0
    - q2-taxa=2023.2.0
    - q2-types=2023.2.0
    - q2-vsearch=2023.2.0

Installing QIIME2 using a bash script

conda activate base
wget https://data.qiime2.org/distro/core/qiime2-2023.2-py38-osx-conda.yml
conda env create -n qiime2-2023.2 --file qiime2-2023.2-py38-osx-conda.yml
conda activate qiime2-2023.2 
qiime info

Download a qiime2 trained classifer

wget \
  -O "gg-13-8-99-515-806-nb-classifier.qza" \
  "https://data.qiime2.org/2023.2/common/gg-13-8-99-515-806-nb-classifier.qza"

Other classifiers also exist. Check on QIIME2 website for more information.



References

[1]
Buza, T. M., Tonui, T., Stomeo, F., Tiambo, C., Katani, R., Schilling, M., … Kapur, V. (2019). iMAP: An integrated bioinformatics and visualization pipeline for microbiome data analysis. BMC Bioinformatics, 20. https://doi.org/10.1186/S12859-019-2965-4


Appendix

Project main tree

.
├── LICENSE.md
├── README.md
├── REFER
│   ├── dags
│   ├── data
│   ├── envs
│   ├── images
│   ├── imap-bioinformatics-qiime2
│   ├── imap-bioinformatics.Rproj
│   ├── index.html
│   ├── render.R
│   ├── report.html
│   ├── resources
│   ├── results
│   ├── rules_dag.sh
│   ├── smk_html_report.sh
│   └── workflow
├── config
│   ├── config.yml
│   ├── pbs-torque
│   ├── samples.tsv
│   ├── slurm
│   └── units.tsv
├── dags
│   ├── rulegraph.png
│   └── rulegraph.svg
├── data
│   ├── README.md
│   ├── logs
│   ├── metadata
│   ├── mothur
│   ├── qiime
│   ├── reads
│   ├── references
│   └── test
├── images
│   ├── bioinformatics.png
│   ├── bkgd.png
│   ├── imap_part02.svg
│   ├── imap_part03.svg
│   ├── imap_part04.svg
│   ├── imap_part05.svg
│   ├── silvaalign.png
│   ├── smkreport
│   └── sra_config_cache.png
├── index.Rmd
├── library
│   ├── apa.csl
│   ├── export.bib
│   ├── imap.bib
│   └── references.bib
├── mothur_process
│   ├── control.final.shared
│   ├── current_files.summary
│   ├── error_analysis
│   ├── final.count_table
│   ├── final.list
│   ├── final.rep.fasta
│   ├── final.sensspec
│   ├── final.steps
│   ├── final.tax.summary
│   ├── intermediate
│   ├── logs
│   ├── mock.final.shared
│   ├── sample.final.0.03.subsample.shared
│   ├── sample.final.braycurtis.0.03.lt.ave.dist
│   ├── sample.final.braycurtis.0.03.lt.dist
│   ├── sample.final.braycurtis.0.03.lt.nmds.axes
│   ├── sample.final.braycurtis.0.03.lt.nmds.iters
│   ├── sample.final.braycurtis.0.03.lt.nmds.stress
│   ├── sample.final.braycurtis.0.03.lt.pcoa.axes
│   ├── sample.final.braycurtis.0.03.lt.pcoa.loadings
│   ├── sample.final.braycurtis.0.03.lt.std.dist
│   ├── sample.final.braycurtis.0.03.lt.tre
│   ├── sample.final.count.summary
│   ├── sample.final.groups.ave-std.summary
│   ├── sample.final.groups.rarefaction
│   ├── sample.final.groups.summary
│   ├── sample.final.shared
│   ├── sample.final.sharedsobs.0.03.lt.ave.dist
│   ├── sample.final.sharedsobs.0.03.lt.dist
│   ├── sample.final.sharedsobs.0.03.lt.std.dist
│   ├── sample.final.thetayc.0.03.lt.ave.dist
│   ├── sample.final.thetayc.0.03.lt.dist
│   ├── sample.final.thetayc.0.03.lt.std.dist
│   ├── test.contigs_report
│   ├── test.files
│   ├── test.scrap.contigs.fasta
│   ├── test.scrap.contigs.qual
│   ├── test.trim.contigs.fasta
│   └── test.trim.contigs.qual
├── qiime2_process
│   ├── demux.qza
│   ├── demux.qzv
│   ├── feature-table.qza
│   ├── feature-table.qzv
│   ├── q2-sample-metadata.qzv
│   ├── rep-seqs.qza
│   ├── rep-seqs.qzv
│   ├── stats.qza
│   └── stats.qzv
├── report.html
├── resources
│   ├── metadata
│   ├── reads
│   └── test
├── results
│   └── project_tree.txt
├── smk.css
├── styles.css
├── tree.sh
└── workflow
    ├── Snakefile
    ├── envs
    ├── report
    ├── rules
    └── scripts

39 directories, 78 files

Static snakemake report

The interactive snakemake HTML report can be viewed by opening the report.html using any compatible browser. You will be able to explore the workflow and the associated statistics. You can close the left bar to get a more expansive display view.

Summary of Informative workflows








Troubleshooting of FAQs

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      2. Answer
  2. Question
      3. Answer